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BRIGITTE SCHLEGELBERGER et al.
Hybridization and post-hybridization washes
Hybridization 1. Apply 1.5 III of the respective hybridization solution to the area of the
slide containing the cells.
2. Cover with a round lO-mm coverslip.
3. Seal with rubber cement.
4. Place the slides at the bottom of a metal box on a wet paper towel, close
the box and denature for 7 min in a water bath at 75°e.
5. Immediately transfer the hot metal box into a 37°C incubator. According to the probe, hybridize from 1 h to 3 days.
Post-hybridization
washes
Preparation
of slides
1. Prewarm three Coplin jars containing 0.1 x SSC at 60°C in a water bath.
2. Remove rubber cement from the slides with a needle.
3. Shake slides in the first Coplin jar at 60°C in 0.1 xSSC until all coverslips are removed.
4. Wash slides by shaking for 5 min at 60°C in each of the three Coplin
jars containing 0.1 x SSe.
5. Equilibrate slides in PN buffer for 2 min at RT.
Simultaneous detection of biotin- and digoxigenin-Iabeled probes with AMCA (blue)
and FllC (green)
The following protocol allows the simultaneous detection of biotinylated
probes with the blue fluorochrome AMCA and digoxigenin-labeled probes
with the green fluorochrome FITe. In analogy, a series of alternative cascades allowing the detection of indirectly labeled probes by other colors or
fluorescent dyes can be designed. The respective solutions can be incubated under coverslips followed by three washings for 2 min each in
PN buffer in a Coplin jar. Nevertheless, the use of Shandon holders
not only facilitates manual detection but also provides the prerequisite
for automatization with pipette robots (e.g. Tecan SLT).
1. Incubate slides with a mixture of Cy3-conjugated-avidin (1:400 in PNM
buffer) and mouse anti-digoxigenin antibody (1:100 in PNM buffer) at
RT for 30 min.
BRIGITTE SCHLEGELBERGER et al.
Hybridization and post-hybridization washes
Hybridization 1. Apply 1.5 III of the respective hybridization solution to the area of the
slide containing the cells.
2. Cover with a round lO-mm coverslip.
3. Seal with rubber cement.
4. Place the slides at the bottom of a metal box on a wet paper towel, close
the box and denature for 7 min in a water bath at 75°e.
5. Immediately transfer the hot metal box into a 37°C incubator. According to the probe, hybridize from 1 h to 3 days.
Post-hybridization
washes
Preparation
of slides
1. Prewarm three Coplin jars containing 0.1 x SSC at 60°C in a water bath.
2. Remove rubber cement from the slides with a needle.
3. Shake slides in the first Coplin jar at 60°C in 0.1 xSSC until all coverslips are removed.
4. Wash slides by shaking for 5 min at 60°C in each of the three Coplin
jars containing 0.1 x SSe.
5. Equilibrate slides in PN buffer for 2 min at RT.
Simultaneous detection of biotin- and digoxigenin-Iabeled probes with AMCA (blue)
and FllC (green)
The following protocol allows the simultaneous detection of biotinylated
probes with the blue fluorochrome AMCA and digoxigenin-labeled probes
with the green fluorochrome FITe. In analogy, a series of alternative cascades allowing the detection of indirectly labeled probes by other colors or
fluorescent dyes can be designed. The respective solutions can be incubated under coverslips followed by three washings for 2 min each in
PN buffer in a Coplin jar. Nevertheless, the use of Shandon holders
not only facilitates manual detection but also provides the prerequisite
for automatization with pipette robots (e.g. Tecan SLT).
1. Incubate slides with a mixture of Cy3-conjugated-avidin (1:400 in PNM
buffer) and mouse anti-digoxigenin antibody (1:100 in PNM buffer) at
RT for 30 min.